Sequence comparisons of plasmids pBJS-O of Spiroplasma citri and pSKU146 of S. kunkelii: implications for plasmid evolution

Background Spiroplasma citri BR3-3X and S. kunkelii CR2-3X cause serious diseases worldwide on citrus and maize species, respectively. S. citri BR3-3X harbors a plasmid, pBJS-Original (pBJS-O), that encodes the spiroplasma adhesion related protein 1 (SARP1), a protein implicated in binding of the pathogen to cells of its leafhopper vector, Circulifer tenellus. The S. kunkelii CR2-3X plasmid, pSKU146, encodes a homolog of SARP1, Sk-ARP1. Due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well. Results The nucleotide sequence of pBJS-O was determined and compared to the sequences of a plasmid from BR3-T (pBJS-T), which is a multiply passaged leafhopper transmissible derivative of BR3-3X, and to known plasmid sequences including that of pSKU146. In addition to arp1, the 13,374 bp pBJS-O sequence putatively contains nine genes, recognized as open reading frames (ORFs). Several pBJS-O ORFs have homologs on pSKU146. However, the sequences flanking soj-like genes on both plasmids were found to be more distant from one another than sequences in any other region. Further, unlike pSKU146, pBJS-O lacks the conserved oriT region characteristic of the IncP group of bacterial plasmids. We were unable to identify a region in pBJS-O resembling a known plasmid origin of transfer. In regions where sequence was available for the plasmid from both BR3-3X and BR3-T, the pBJS-T sequence had a 0.4 kb deletion relative to its progenitor, pBJS-O. Southern blot hybridization of extrachromosomal DNA from various S. citri strains and spiroplasma species to an arp-specific probe and a probe made from the entire plasmid DNA of BR3-3X revealed limited conservation of both sequences in the genus Spiroplasma. Finally, we also report the presence on the BR3-3X chromosome of arp2, an S. citri homolog of arp1 that encodes the predicted protein SARP2. The C-terminal domain of SARP2 is homologous to that of SARP1, but its N-terminal domain is distinct. Conclusion Our data suggest that pBJS is a novel S. citri plasmid that does not belong to any known plasmid incompatibility group. The differences between pBJS-O and pSKU146 suggest that one or more events of recombination have contributed to the divergence of the plasmids of the two sister Spiroplasma species; the plasmid from S. citri itself has diverged slightly during the derivation of S. citri BR3-T from BR3-3X. Our data also show that pBJS-O encodes the putative adhesin SARP1. The presence of traE and mob on pBJS-O suggests a role for the plasmid in spiroplasmal conjugation.


Background
The phytopathogenic spiroplasmas and phytoplasmas, which cause serious diseases of economically important plant species worldwide [ [1] and [2]], are wall-less prokaryotes phylogenetically related to Gram-positive eubacteria with low G+C content [3]. They are transmitted in nature by phloem-feeding insects, predominantly leafhoppers, in a propagative manner [4]. Even though there are close to forty recognized spiroplasma species, only three plant pathogenic spiroplasmas have been identified and characterized to date. S. citri [[5,6] and [7]] is the causative agent of stubborn disease of citrus and brittle root disease of horseradish; S. kunkelii [[8,9] and [10]] is the etiological agent of corn stunt; and S. phoeniceum [11] causes periwinkle yellows. Unlike phytoplasmas, spiroplasmas can be cultured in vitro. Therefore, the relationships between S. citri and its insect vectors, the beet leafhopper, Circulifer tenellus, and the related species, C. haematoceps [12], have been investigated extensively, serving as models for investigating the molecular aspects of mollicute-vector interactions.
Spiroplasma binding to insect host and non-host cells, both in tissue-culture and within the intact insect, has been reported [ [13] and [14]]. The loss and restoration of the ability of S. citri to adhere to tissue-cultured C. tenellus cells was associated with degradation and restoration of P89 (designated SARP1), a spiroplasma membrane protein [14]. Due to the possible direct involvement of SARP1 in the spiroplasma-leafhopper interaction, it was hypothesized that SARP1 is an adhesin. Later, Berg et al [15] reported cloning and characterization of arp1, the gene encoding SARP1, from S. citri BR3-T. They also reported that mature SARP1 protein contains a novel domain at the N-terminus, called "sarpin", made of six repeats of 39-42 amino acids each.
S. citri harbors several extrachromosomal DNAs with unique restriction patterns [ [16][17][18] and [19]]. S. citri lines, derived from a clone, and sister clones of the same lines showed differences in their extrachromosomal DNAs [20]. In addition to known plasmids, there are replicative forms (RFs) of several viruses and other uncharacterized circular extrachromosomal DNAs in S. citri [21].
Plasmids have also been noted in strains of S. kunkelii [22]. Recently, Davis and colleagues [23] reported the complete sequence of the S. kunkelii CR2-3X plasmid pSKU146, which encodes a homolog of SARP1, Sk-ARP1. In the present study, we isolated and characterized a related indigenous plasmid, designated pBJS-Original (pBJS-O), from S. citri BR3-3X. This is a report of the discovery, distribution and characterization of that plasmid. Among other genes, pBJS-O contains arp1. The significance of the discovery of pBJS-O in relation to our current understanding of the S. citri-leafhopper interactions and potential genetic manipulations in mollicutes is discussed. Implications for the evolution of both pBJS-O and pSKU146 are also presented.

Results
Detection and analysis of arp2 SARP1 has been characterized previously and the gene encoding it, arp1, has been cloned and sequenced [Gen-Bank:AJ297706] from S. citri BR3-T [15]. In the process, an RsaI restriction fragment was cloned and sequenced from BR3-T genomic DNA; the alignment of this fragment with AJ297706 revealed 92% similarity in the 3' 660 nucleotides of the former sequence. However in the 5' 55 bases of the total 715 bp, upstream from position 2370 in AJ297706, the new fragment was not similar to the known sequence. We designated this gene, which resembles but is not identical to arp1, as arp2 and its putative protein product as SARP2. As also noted by Bai et al. [22], the S. kunkelii CR2-3X genome contains two sequences similar to those of S. citri BR3-T arp genes. The predicted protein, Sk-ARP1 (for S. kunkelii adhesion related protein 1), encoded by the first sequence, Sk-arp1, contains seven rather than six sarpin repeats and has C-terminal domains resembling those of SARP1 [15]. The second sequence encodes a putative protein whose C-terminus is homologous to that of SARP1, but has an unrelated N-terminus. This protein is designated Sk-ARP2 (S. kunkelii adhesion related protein 2) and the corresponding gene is named Sk-arp2. SARP1 has sequence similarity with known adhesins. Fleury et al. Southern blotting hybridization of (A) S. citri BR3-3X, BR3-G, BR3-T, and (B) S. citri BR3-M and BR3-P plasmid preparations to an arp1-derived probe Figure 2 Southern blotting hybridization of (A) S. citri BR3-3X, BR3-G, BR3-T, and (B) S. citri BR3-M and BR3-P plasmid preparations to an arp1-derived probe. EcoRI-digested S. citri BR3-3X chromosomal DNA and EcoRI-digested and undigested plasmid preparations from BR3-3X, BR3-G, BR3-T, BR3-M and BR3-P are shown. D, digested with EcoRI; U, undigested. Hybridization in the marker lane is due to presence of short pBluescript vector sequences in the probe. [24] have shown that the predicted amino acid sequence of P40, a Mycoplasma agalactiae cytadhesin, is similar not only to that of SARP1 but also to the one of P50, an adhesin of M. hominis.

Isolation and distribution of Spiroplasma extrachromosomal DNA
We isolated extrachromosomal DNA from S. citri BR3-3X to test the hypothesis that this DNA contains an arp-like gene as in S. kunkelii. Restriction of the DNA with single enzymes, including BglII and NdeI, converted a DNA migrating with 9 kb into a fragment migrating close to 7 kb (Fig. 1). These results were consistent with the presence of a single major plasmid. We designated the plasmid pBJS-O. By nucleotide sequencing, we determined that the actual size of the plasmid was 13,374 bp and deposited the sequence in the EMBL Nucleotide Sequence Database [EMBL:AJ972409] To test the conservation of pBJS-O in S. citri strains derived from S. citri BR3, plasmid preparations from S. citri BR3-3X and from BR3-G, BR3-T, BR3-M and BR3-P, lines derived from BR3-3X, were probed with a DNA fragment derived from arp1 ( Fig. 2 and Table 1). All hybridized with the probe, producing two or more bands. To test the conservation of pBJS-O in other S. citri strains, other plant-associated spiroplasmas and the closest relative of S. citri, S. melliferum [25], the plasmids of S. kunkelii CR2-3X, S. melliferum, S. citri strains R8A2, ASP-1 and Beni Mellal, S. floricola and S. phoeniceum also were probed with the arp1-derived probe (Fig. 3A). Only S. kunkelii CR2-3X and S. melliferum reacted in the hybridization. However, when the same plasmids were probed with the whole pBJS-O plasmid as a probe (Fig. 3B), all the sample preparations, except those from S. citri Beni Mellal, S. floricola and S. phoeniceum, hybridized with the probe. All of the above Southern hybridization experiments revealed multiple reactive species in the plasmid preparations and the hybridization patterns of EcoRI-digested and undigested plasmid samples were very similar to each other. For comparison, the blots included EcoRI-digested chromosomal DNA of S. citri BR3-3X. A single hybridization signal distinct from those of plasmid preparations was observed (Fig. 2). The Southern blot hybridization results suggest that arprelated sequences are present on both a plasmid and the chromosome. arp1 and arp2 from BR3-T are nearly identical over a considerable portion of their nucleotide sequence. Hence, using a probe containing this conserved region should detect both genes. Nevertheless, arp1 and arp2 differ at several positions in those regions. To determine whether the BR3-3X plasmid and chromosomal sequences represented arp1 or arp2 genes, we determined parts of the sequences of BR3-3X plasmid and chromosomal DNAs by direct sequencing and by sequencing amplified PCR products. Comparison of the BR3-3X arp sequences with those of BR3-T revealed that the BR3-3X arp2 sequence had diverged more from the other three sequences than the latter had from each other (Figs. 4 and 5). At positions where the two BR3-T genes differed from one another, the chromosomal BR3-3X sequence had arp2 residues in 21 positions and arp1 residues in only 3 positions (Figs. 4A and 4B). Conversely, at arp1-and arp2-specific positions, the BR3-3X plasmid DNA had no arp2 residues and 28 arp1 residues. Further, at all 57 positions at which chromosomal and plasmid sequences differed, the BR3-3X plasmid and arp1 nucleotides were identical. Hence, we conclude that, in S. citri BR3-3X, the arp1 gene resides on a plasmid and that the arp2 gene most likely resides on the chromosome. The newly determined arp2 sequences from BR3-3X and BR3-T were deposited in the EMBL Nucleotide Sequence Database [EMBL:AM040506 and EMBL:AM040505, respectively].

Complete pBJS-O sequencing and analysis
The 4273 bp sequence [GenBank:AJ297706] originally cloned and characterized from S. citri BR3-T [15] contains a partial ORF soj, followed by ORF2, P89 (arp1) and another partial ORF, ORF4. AJ297706 was used to design primers and initiate primer walking to determine the complete pBJS-O plasmid sequence and allow its characterization. During sequencing, a segment (from nucleotide 1-80) of the assembled sequence proved particularly difficult to sequence. It contained three of the six oligopurine/oligopyrimidine tracts of 12 or more bp in the entire plasmid sequence. That the sequence of the tracts was consistent with triple-helix formation suggests that this region of the plasmid may readily form triplehelical structures interfering with sequencing.
The total plasmid sequence is 13,374 bp in length and contains ten predicted ORFs ( Fig. 6 and  An unrooted phylogenetic tree representing the nucleotide sequence alignment shown in Fig. 4A. The tree was generated using algorithms ClustalW and PHYLIP from the Biology Workbench using the neighbour-joining method. Comparison of partial nucleotide sequences of S. citri BR3-3X chromosomal (BR3-3X_chr) and plasmid (BR3-3X_pl) sequences to those of available BR3-T arp1 and arp2 genes (BR3-T_arp1 and BR3-T_arp2, respectively) Algorithm TMHMM v. 2.0 was used to predict the locations of transmembrane helices and intervening loops in the putative products of traE (Fig. 7), mob and orf4. Although the TraE polypeptide was predicted to contain three transmembrane helices, the third helix was predicted at a lower probability than were the other two.
Assuming the presence of three transmembrane helices, the protein was predicted to have the N-terminal region (about 10% of the length of the polypeptide) in the cytosol and almost all of the rest of the protein extracellular.
Plasmid pSKU146 from S. kunkelii CR2-3X encodes the S. kunkelii homolog of SARP1, Sk-ARP1. In addition to sk-arp1, pSKU146 contains 17 ORFs. The pSKU146-ORFs having counterparts on pBJS-O were listed above. However, although both plasmids contain genes encoding the ParA-like protein, Soj, sequences surrounding those genes are more distant from one another than are sequences in any other regions. Further, unlike pSKU146, pBJS-O lacks the conserved oriT region characteristic of the IncP group of bacterial plasmids. Also, we were unable to identify a region in pBJS-O resembling a known plasmid origin of transfer.

Discussion
In the present study we report isolation, distribution and structural characterization of pBJS-O, an indigenous S. citri BR3-3X plasmid. We also present evidence that pBJS-O harbors arp1, the gene encoding SARP1, and describe the presence on the BR3-3X chromosome of arp2, an S. citri homolog of arp1. Finally, the sequences of pBJS-O, pBJS-T and the S. kunkelii CR2-3X plasmid, pSKU146, in relation to plasmid evolution are discussed.

Conservation of arp and pBJS-O sequences in Spiroplasma
In Southern hybridizations, the similarity in the hybridization patterns of EcoRI digested versus undigested pBJS-O preparations, despite the presence of two GAATTC recognition sequences, may be due to an adenine methylation system in S. citri. Restriction site modification in S. citri has been reported elsewhere. Rascoe et al. [26] detected multiple bands of S. citri extrachromosomal DNA by Southern blotting, which they attributed to incomplete restriction due to variable restriction site modification in the DNA, and Ye et al. [27] reported protection of an EcoRI site in the S. citri 16S rDNA. Moreover, differential methylation of restriction sites in the RF of the spiroplasma virus, SVTS2, allowed Sha et al. [28] to clone the full-length DNA.
S. citri BR3-3X showed probe-reactive sequences in both the chromosomal and extrachromosomal DNA fractions. However, that the patterns of hybridization of the two fractions differed significantly from each other demonstrates that the two fractions of BR3-3X DNA were not appreciably cross-contaminated. Sequence analyses of DNA from the two fractions showed that, in BR3-3X, arp1 resides on pBJS-O and arp2 on the chromosome. Hybridization of S. citri ASP-1 and R8A2 plasmid preparations with the pBJS-O probe (Fig. 3B), but not with the arp1 probe (Fig. 3A), indicates that each of these two strains contained a plasmid related to pBJS-O, which differed from pBJS-O in lacking arp1. Although S. citri ASP-1 and R8A2 were originally derived from the same parent strain, both have undergone extensive cultivation in vitro since their first isolation, which may have contributed to the differences between their plasmids and pBJS-O. Moreover, the differences in the maintenance regimes of the various spiroplasmas tested may have contributed to the evolution of their plasmids. In this paper we could not correlate pBJS-O and pBJS-O like sequences with either transmissibility or phytopathogenicity of the spiroplasmas tested. However, it is still hypothesized that SARP1 is involved in S. citri transmission by the insect vector.
Frequent chromosomal rearrangements such as inversions and deletions, leading to genome instability, have been reported in spiroplasmas, such as in the lines derived The ORF and restriction map of pBJS-O Figure 6 The ORF and restriction map of pBJS-O.  [30]]. In the present study, we detected a 0.4 kbp deletion in pBJS-T relative to pBJS-O. Unlike BR3-3X, which was stored frozen, S. citri BR3-T was maintained for several years in turnip plants via transmission by the natural insect vector C. tenellus, possibly leading to the sequence differences between pBJS-T and pBJS-O. A recombinational chromosomal rearrangement is indicated by the 5'-sequence differences between arp1 and arp2 reported above.
Recombination likely also played a role in the generation of pBJS-O like plasmids. The gene organization on pBJS-O is similar to that of the recently characterized IncP-like S. kunkelii CR2-3X plasmid, pSKU146. Yet, the two plasmids have substantially different sequences in the region including the soj-like gene in both plasmids and the IncP oriT sequence in pSKU146. Highly similar sequences in the remainder of the two plasmids suggest that recombination events have occurred during the generation of one or both plasmids.
Phage sequences have been implicated in many recombination events in spiroplasmas. Only a short region with similarity to a phage gene was found in pBJS-O. However, the observation of strong stops to sequencing reactions in the region of nucleotides 1 to 80 is reminiscent of a strong stop encountered during the sequencing of the SVTS2 phage [31]. This strong stop was attributed to potential secondary structure putatively involved in phage packaging. It is, thus, possible that pBJS-O has some phage-like properties.

pBJS-O genes
As mentioned above, ORF3, encoding SARP1, and adjacent ORFs [GenBank:AJ297706], had been cloned and characterized from S. citri BR3-T [15]. ORF3 was flanked downstream by a partial ORF (ORF4) having no known homologs. Upstream, ORF3 was flanked by ORF2, encoding a hypothetical protein with no similarity to any known protein, and ORF1, a partial ORF encoding a putative homolog of a ParA-like protein, Soj, which oscillates from pole to pole [32] and is important for chromosome partitioning in Bacillus subtilis [33]. In this study, the putative protein product of orf4 was predicted to contain eight transmembrane helices. Due to a 0.4 kb deletion in the derivation of pBJS-T, orf4 is possibly a part of the same transcription unit as arp1 and traE in this strain. In BR3-3X, arp1 and orf4 are separated by 281 bp, suggesting that they are transcribed separately. Consistent with different translational constraints on this region in BR3-T and BR3-3X, this region contains a large proportion of the differences between the lines. The translation start site of traE was predicted to be ten nucleotides upstream of the orf4 translation stop site.
Consistent with the observation of Bai et al. [22], putative products of the other pBJS-O ORFs, traE [34] and mob [35], are homologous to proteins that are components of the bacterial type IV secretion system involved in conjugative DNA transfer. Members of the TraE family of proteins are thought to form pili that, in addition to conjugation, are involved in processes like virus infection and biofilm formation. Bai et al. [22] reported the presence of three conserved transmembrane helices in four TraE homologs that they characterized from S. kunkelii M2.
Ozbek et al. [36], in their transmission electron micrographs, reported the presence of structures resembling fimbriae and pili in S. kunkelii and Bai et al. [22] considered whether the structures may be involved in conjugation. Bové [37] reported that rod-shaped spiroplasma viruses, approximately 230-280 by 10-15 µm in size, can also be surface-associated. Because they can attach perpendicularly to the host membrane at their tips [ [38] and [39]], they might resemble the structures reported as pili/fimbriae. In the putative TraE homolog reported here, unlike its S. kunkelii counterpart, two transmembrane helices were predicted at high probability and a third one at moderate probability. Should the third not actually be a transmembrane helix, the ATP binding site would be located intracellularly rather than extracellularly.

pBJS-O gene organization and evolution
Unlike pSKU146, pBJS-O was found to lack the conserved oriT region characteristic of the IncP group of plasmids. We were also unable to identify a region in pBJS-O resembling any other known plasmid origins of transfer, suggesting that pBJS-O belongs to a hitherto unidentified group of plasmids. Horizontal transfer of a promiscuous plasmid, such as an IncP plasmid, between phylogenetically related and unrelated bacteria would help the hosts quickly adapt to different niches [40]. It is possible that an IncP-like plasmid was acquired by the ancestor of S. citri and S. kunkelii. The plasmid may have co-evolved with the host chromosomes after the divergence of the two species, leading to the emergence of pBJS-O and pSKU146, respectively, and to the adaptation of the pathogens to phylogenetically distinct leafhopper vectors and plant hosts.

Future directions
Molecular genetic tools such as cloning and transposonmediated mutagenesis are available for the study of mollicutes [41]. Cloned genes were expressed in S. citri GII-3 using artificial plasmids based on the S. citri chromosomal oriC [ [42,43] and [44]]. However, those plasmids tend to integrate into the S. citri chromosome. When pCJ32, a derivative of the oriC plasmid pBOT1, containing an internal fragment of the gene scm1 (a motility-related S. citri gene), was transformed into S. citri GII-3 cells it successfully integrated into the host chromosome by homologous recombination and disrupted scm1, resulting in non-motile S. citri GII-3 mutants [45]. However, attempts to use pBOT1 in S. citri BR3-3X have been unsuccessful (F. Ye, unpublished data), possibly due to the incompatibility of the plasmid with the host. The indigenous S. citri BR-3X plasmid, pBJS-O, will help us develop a better vector for genetic manipulation not only in S. citri BR3-3X but also in other spiroplasmas.

Conclusion
We have shown that the S. citri BR3-3X plasmid, pBJS-O, encodes the putative adhesin SARP1. This is the first report of an S. citri plasmid encoding a putative adhesin.
We have further shown that the arp1-like gene, arp2, resides on the BR3-3X chromosome. The indigenous S. citri BR3-3X plasmid, pBJS-O, will be useful for the development of a better vector for genetic manipulation not only in S. citri BR3-3X but also in other spiroplasmas. Our data also suggest that pBJS-O is a novel S. citri plasmid that does not belong to any known plasmid incompatibility group. The differences between pBJS-O and pSKU146 suggest that recombination has contributed to the divergence of the two plasmids.

Purification of chromosomal and extrachromosomal ds DNAs from spiroplasmas
For Southern blot hybridization and PCR, extrachromosomal double-stranded (ds) DNA of spiroplasma strains was isolated using the QIAprep Spin Miniprep and the QIAGEN Plasmid Mini Kits (Qiagen, Santa Clarita, CA), following the manufacturer's protocols. For primer walking, S. citri BR3-3X extrachromosomal DNA was isolated using a previously published procedure [28]. The isolation of chromosomal DNA from S. citri BR3-3X cells was performed according to Murray  Hybridizations were performed at 55°C in Church buffer (0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, and 1 mM EDTA) overnight followed by four washes, each of 20 min, at 55°C in washing buffer (40 mM sodium phosphate buffer, pH 7.2, containing 0.1% SDS). Detection of the DIG-labeled probes was performed using a DIG Luminescent Detection Kit (Roche) following the manufacturer's protocol.

Complete nucleotide sequencing of pBJS-O
The sequence AJ297706 was used to design primers to initiate primer walking to completely sequence and characterize the unknown portion of pBJS-O. The sequencing reactions were performed using ~ 1.2 µg of pBJS-O DNA and 40 µM of primers with the ABI PRISM BigDye Terminator Cycle Sequencing method and the ABI PRISM 3700 Automated DNA Analyzer, as mentioned above. The total 134 sequence reads with an average length of 600 bases gave us about 6× coverage of the entire plasmid sequence. The fragments were assembled from the trace files using the software package PipeOnline 2.0 [53]. Physical gaps in the sequence were closed by PCR and cloning of the products into vector pGEM-T (Promega). The clones were sequenced using primers T7 and SP6. The consensus sequence of the final assembly was annotated using the BLASTX search program [54] and the ORF Finder tool at NCBI, in which a minimum length of 100 bases was used for the nucleotide sequence of a putative ORF. The nucleotide and amino acid sequence analysis tools offered by the Biology Workbench at the San Diego Supercomputer Center, such as ClustalW and PHYLIP for generating the unrooted phylogenetic tree of the S. citri arp sequences, were used to further analyze the plasmid and the polypeptide sequences. BLASTN and BLASTP searches were carried out to find out relationships with the closest homologs. S. kunkelii CR2-3X genome sequence data were accessed and BLAST searches were performed at the Spiroplasma Genome Sequencing Project Web site mentioned above.